Annexins are a family of proteins that provide a regulated link between Ca2+ signaling and a number of membrane-related functions including ion channel activity, apoptosis, membrane fusion and domain organization. The functional hallmark of annexins is Ca2+-dependent binding to phospholipid bilayers but recent studies also have detected Ca2+-independent binding. Using the interaction of annexin B12 with phospholipid vesicles as a model system, we undertook an extensive study designed to provide the structural and dynamic information needed to define the biophysical mechanism by which annexins interact with membranes and to evaluate proposed biological functions. Our studies showed that annexin B12 exists in three structural forms: a soluble monomer, a Ca2+-dependent peripheral membrane-bound trimer, and a Ca2+-independent transmembrane channel that forms at mildly acidic pH. These three forms undergo reversible inter-conversion with the equilibrium being modulated by phospholipid, Ca2+ and H+. The backbone fold of the Ca2+-dependent membrane-bound annexin B12 was nearly identical to that of our crystal structure of the soluble protein while transmembrane insertion involved global "inside-out" refolding. Experiments in this proposal mainly focus on the structure and biological function of the novel transmembrane form of annexins although some effort will be directed toward investigating the biophysical implications of our previous studies of the Ca2+-dependent membrane-bound trimer. To optimize our opportunities to directly connect our in vitro structural studies to biological function, we propose to extend our studies of hydra annexin B12 to a protein that appears to be its homologue, human annexin A5. The goals of this project are to determine a high resolution structure for the transmembrane form of annexins, to investigate the role of membrane curvature (or curvature stress) in determining annexin structure/function and to explore the biological implications of these studies by investigating the role of annexin A5 in the mitochondrial apoptotic pathway. The experimental techniques used in these studies will be site-directed spin labeling, fluorescence spectroscopy, fluorescence microscopy and other biochemical and biophysical methods. Since annexins have been implicated in human diseases including cancer, viral infection, fibrinolysis, and blood coagulation, a more complete understanding of their structure and function may identify therapeutic targets. [unreadable] [unreadable] [unreadable]